Methods and compositions for inducing autoimmunity in the treatment of cancers

ABSTRACT

Disclosed are methods and compositions for the prevention and treatment of cancers using lipid-carrier protein conjugate compositions for generating lipid-specific immune responses in an animal. Also disclosed are methods for making phosphatidylserine conjugate compositions and their formulation for use in a variety of pharmaceutical applications including the detection and treatment of a variety of cancers and related conditions.

This application is a division of Ser. No. 09/224,558 filed Dec. 30,1998 now U.S. Pat. No. 6,300,308.

The present application is a continuation-in-part of U.S. ProvisionalPatent Application Serial No. 60/071,794 filed Dec. 31, 1997. The entiretext of the above-referenced disclosure is specifically incorporated byreference herein without disclaimer.

The United States has certain rights in the present invention pursuantto Grant DK41714 from the National Institutes of Health.

1.0 BACKGROUND OF THE INVENTION

1.1 Field of the Invention

The present invention relates generally to the field of oncology. Moreparticularly, certain embodiments concern methods for making and usinglipid-carrier protein conjugate compositions such as phosphatidylserine(PS)-conjugates for generating lipid-specific immune responses in ananimal. Also disclosed are methods for making PS antigen and antibodycompositions and their use in a variety of therapeutic applications,including the formulation of pharmaceutical compositions for theprevention and treatment of cancers.

1.2 Description of the Related Art

The results of many studies have led to the concept that membranephospholipid asymmetry is ubiquitous. The outer leaflet of eukaryoticplasma membranes contains most of the cholinephospholipids, whereas theaminophospholipids are mainly present in the cell's inner leaflet(Devaux, 1991; Schroit and Zwaal, 1991). While asymmetry seems to be therule for normal cells, loss of membrane lipid sidedness, in particularthe emergence of phosphatidylserine (PS) at the cell surface, results inthe expression of altered surface properties that modulates cellfunction and influences the cells interaction with its environment(Zwaal and Schroit, 1997). For example, the exposure of PS promotescoagulation and thrombosis by platelets (Bevers et al., 1983; Rosing etal., 1985; Thiagarajan and Tait, 1990) and the recognition of apoptotic(Fadok et al., 1992; Bennett et al., 1995; Sambrano and Steinberg, 1995;Verhoven et al., 1995) and aged (Herrmann and Devaux, 1990; Geldwerth etal., 1993; Connor et al. 1994) cells by the reticuloendothelial system.

To characterize these and other PS-related processes, new tools arerequired to determine physiologically-dependent alterations in thedistribution of PS in cell membranes. Although the application ofclassical biochemical methodologies (Gordesky et al., 1975; Schick etal., 1976; Etemadi, 1980; Bevers et al., 1982) has yielded importantinformation on PS asymmetry, most of these methods are invasive anddestructive. Recently developed methods, such as the PS-dependentprothrombinase assay (Bevers et al., 1983; Rosing et al., 1980; VanDieijen et al., 1981) and labeled annexin V binding (Thiagarajan andTait, 1990; Tait and Gibson, 199,4; Vermes et al., 1995; Kuypers et al.,1996), are non-invasive and have provided the means to assess thepresence and topology of PS in the outer leaflet of viable cells. Thesemethods, however, require the inclusion of various plasma cofactorsand/or divalent cations which might influence the lateral distributionof lipids in the plane of the membrane.

While antibodies against different membrane components has become anindispensable aid in the study of membrane structure and function,little attention has been given to the application of lipid-specificantibodies for studying lipid-dependent processes. Because of theinherent difficulty of producing antibodies against small highlyconserved lipids, the development of lipid antibodies has progressedslowly. Nonetheless, several laboratories have produced antibodiesagainst certain phospholipid species by immunization with liposomes(Maneta-Peyret et al., 1988; Maneta-Peyret et al., 1989; Banedi andAlving, 1990) or by adsorption of monomeric phospholipids to proteins(Maneta-Peyret et al., 1989; Tamamura et al., 1971), bacteria (Umeda etal., 1989) and acrylamide (Maneta-Peyret et al., 1988; Maneta-Peyret etal., 1989). Antibodies produced by these methods, however, maycross-react with different lipids (Banedi and Alving, 1990; Umeda etal., 1989) and other phosphate-containing moieties (Alving, 1986).

1.3 Deficiencies in the Prior Art

While some methods have been developed in these areas, what is lackingin the prior art are effective methodologies for generating immuneresponses that are useful in various treatment regimens, including thosespecific for oncology.

Several reports on the production of PS antibodies have been published.These include unrelated methods and approaches using PS-containingliposomes (Banedji and Alving, 1990), PS-coated Salmonella (Umeda etat., 1989) and acrylamide-entrapped PS (Maneta-Peyret et al., 1988).There is one report concerning carrier (KLH)-coupled PS (Bate et al.,1993). However, the chemistry employed to make the conjugate coupleslipid to the protein carrier via the lipids primary amine therebydestroying antigenic specificity. Immunization with this conjugateproduced antibody activity which inhibited the production of tumornecrosis factor by malaria-infected erythrocytes. Whether the antibodieshad any lipid specificity was not investigated. This is unlikely,however, considering that the conjugation chemistry destroyed the keyprimary amine determinant of phosphatidylserine. Thus, there exists animmediate need for an effective method of producing highly-specificanti-PS antibodies and cell-mediated PS responses for use in thediagnosis and treatment of various cancers and related conditions.

2.0 SUMMARY OF THE INVENTION

The present invention overcomes one or more of these and other drawbacksinherent in the prior art by providing novel compositions and methodsfor their use in the induction of an autoimmune response to lipids suchas PS. Disclosed are methods for the preparation and use of novel lipidantigen compositions which generate an immune response in an animal.Also disclosed are methods for the use of lipid-specific antibodycompositions, including those specific for PS, in a variety ofdiagnostic and therapeutic regimens, including the treatment of cancer.

Exemplary preferred methods and compositions according to thisinvention, which will be described in greater detail in the remainder ofthe invention include:

Methods for inhibiting cancer cell growth or killing cancer cells,comprising eliciting an immune response with an immunologicallyeffective amount of a composition comprising aphosphatidylserine/polypeptide conjugate;

Methods for treating cancer comprising eliciting an immune response withan immunologically effective amount of a composition comprising aphosphatidylserine/polypeptide conjugate;

Methods for treating cancer comprising contacting a subject with a lipidor lipid/polypeptide conjugate effective to treat said cancer;

Methods of generating an immune response, comprising administering to ananimal a pharmaceutical composition comprising an immunologicallyeffective amount of a phosphatidylcholine/polypeptide or aphosphatidylserine/polypeptide conjugate composition;

Methods for treating cancer in an animal, comprising generating in saidanimal an immune response to a composition comprising aphosphatidyiserine or phosphatidylserine/polypeptide conjugate effectiveto treat said cancer,

Methods of making an antibody that specifically binds tophosphatidylserine or a phosphatidylcholine/polypeptide or aphosphatidylserine/polypeptide conjugate, said methods comprisingadministering to an animal a pharmaceutical composition comprising animmunologically effective amount of a phosphatidylcholine/polypeptide ora phosphatidylserine/polypeptide conjugate composition. Presentlypreferred conjugates for use in such methods are, for example,phosphatidylserine/BSA, phosphatidyiserine/KLR phosphatidylserine/BGG,and phosphatidylserine/β₂-glycoprotein I conjugate;

Antibodies that specifically bind to phosphatidylserine or aphosphatidylcholine/polypeptide or a phosphatidylserine/polypeptideconjugate, said antibody made by a process comprising administering toan animal a pharmaceutical composition comprising an immunologicallyeffective amount of a phosphatidylcholine/polypeptide or aphosphatidyiserine/polypeptide conjugate composition. Presentlypreferred conjugates for use in such processes are, for example,phosphatidylserine/BSA, phosphatidylserine/KLH, phosphatidylserine/BGG,and phosphatidylserine/β₂-glycoprotein I conjugate;

Methods for detecting a phosphatidylserine,phosphatidylcholine/polypeptide or a phosphatidylserine/polypeptideconjugate in a biological sample, comprising the steps of:

(a) obtaining a biological sample suspected of containing aphosphatidylcholine/polypeptide or a phosphatidylserine/polypeptideconjugate;

(b) contacting said sample with a first antibody that binds to aphosphatidylcholine/polypeptide or a phosphatidylserine/polypeptideconjugate, under conditions effective to allow the formation of immunecomplexes; and

(c) detecting the immune complexes so formed; and

Immunodetection kits comprising, in suitable container means, anantibody that specifically binds to phosphatidylserine or to aphosphatidylserine/polypeptide conjugate, and an immunodetectionreagent.

In an important embodiment, the invention provides antigenic PSconjugate compositions and means for making and using thesecompositions. In the context of this invention, a PS composition isunderstood to comprise one or more phosphatidylserine compositions thatare able to generate an immune response in an animal. A PS antibodycomposition is understood to mean an antibody which is specific for PS.Preferably, the antigen composition comprises a lipid-carrier proteinconjugate. The carrier protein may be maleimide-activated, oralternatively, may be prepared by introduction of reactive sulfhydrylsinto the carrier protein. Alternatively, one may prepare proteins bynon-covalent electrostatic interactions between negatively-chargedanionic phospholipids and lipid binding proteins such as β₂-glycoproteinI, also known as apolipoprotein H. Exemplary carrier proteinscontemplated to be useful in the present methods include variouscommonly used carrier proteins including BSA (bovine serum albumin), KLH(keyhole limpet hemocyanin), BGG (bovine gamma globulin) and diphtheriatoxin. As such, a PS composition of the present invention is alsounderstood to comprise one or more PS-containing or other negativelycharged formulations that elicit an immune response in an animal.

2.1 Lipid-specific Antibody Compositions

In a preferred embodiment, administration of a therapeutically effectivedose of a lipid-conjugate antigen composition, such as a PS-conjugate toan animal induces in the animal antibodies which are specific for theparticular lipid. In one embodiment, the carrier protein is aglycoprotein, such as β₂-glycoprotein I.

In certain aspects, the present invention concerns novel lipid-carrierantigen compositions which evoke a specific immune response to thelipid. In particular, PS antigen compositions have been developed whichhave shown remarkable utility both in vitro and in vivo. In particular,PS antigen compositions have been produced to provide vaccine ortherapeutic compositions useful in the prevention or treatment ofvarious cancers, such as lymphomas and renal and bladder cancers.

2.2 Methods for Generating an Immune Response

A further aspect of the invention is the preparation of immunologicalcompositions comprising both antibody and cell-mediated immune responsesfor diagnostic and therapeutic methods relating to the detection andtreatment of a variety of cancers and related illnesses.

The invention also encompasses PS antigen and antibody compositionstogether with pharmaceutically-acceptable excipients, carriers,diluents, adjuvants, and other components, such as peptides, antigens,or pharmaceuticals, as may be employed in the formulation of particularvaccines or antibody compositions.

Antibodies may be of several types including those raised inheterologous donor animals or human volunteers immunized with PScompositions, monoclonal antibodies (mAbs) resulting from hybridomasderived from fuisions of B cells from PS-immunized animals or humanswith compatible myeloma cell lines, so-called “humanized” mAbs resultingfrom expression of gene fusions of combinatorial determining regions ofmAb-encoding genes from heterologous species with genes encoding humanantibodies, or PS-reactive antibody-containing fractions of plasma fromhuman or animal donors.

Also disclosed is a method of generating an immune response in ananimal. The method generally involves administering to an animal apharmaceutical composition comprising an immunologically effectiveamount of a PS composition disclosed herein Preferred animals includemammals, and particularly humans. Other preferred animals includemurines, bovines, equines, ovines, caprines, opines, porcines, canines,felines, and the like. The composition may include partially orsignificantly purified PS antigen compositions, and particularly willinclude one or more of the PS conjugate compositions described herein.

By “immunologically effective amount” is meant an amount of apeptide/lipid composition that is capable of generating an immuneresponse in the recipient animal. This includes both the generation ofan antibody response (B cell response), and/or the stimulation of acytotoxic immune response (T cell response). The generation of such animmune response will have utility in both the production of usefulbioreagents, e.g., CTLs and, more particularly, reactive antibodies, foruse in diagnostic embodiments, and will also have utility in variousprophylactic and therapeutic embodiments.

Immunoformulations of this invention, whether intended for vaccination,treatment, or for the generation of antibodies useful in the detectionof PS or other lipids may comprise native, or synthetically-derived PSantigenic compositions produced using the methods described herein. Assuch, antigenic functional equivalents of the PS compositions describedherein also fall within the scope of the present invention. An“antigenically functional equivalent” protein or peptide is one thatincorporates an epitope that is immunologically cross-reactive with oneor more epitopes derived from any of the particular PS compositionsdisclosed herein. Antigenically functional equivalents, or epitopicsequences and lipid formulations, may be first designed or predicted andthen tested, or may simply be directly tested for cross-reactivity. Alsoencompassed by the invention are modified PS-conjugates which haveimproved antigenicity or other desirable characteristics, and that areproduced in a fashion similar to those described herein.

In still further embodiments, the present invention concernsimmunodetection methods and associated kits. It is contemplated that thePS antigen compositions, and particularly PS conjugates, may be employedto detect antibodies having reactivity therewith, or, alternatively,antibodies prepared in accordance with the present invention, may beemployed to detect PS-containing cells, compositions, tissues, and thelike. Either type of kit may be used in the immunodetection ofcompounds, present within clinical samples. The kits may also be used inantigen or antibody purification, as appropriate.

In general, the preferred immunodetection methods will include firstobtaining a sample suspected of containing a lipid-specific antibody,such as a biological sample from a patient, and contacting the samplewith a first lipid and/or lipid conjugate antigen composition underconditions effective to allow the formation of an immunocomplex (primaryimmune complex). One then detects the presence of any primaryimmnunocomplexes that are formed.

Contacting the chosen sample with the lipid antigen composition underconditions effective to allow the formation of (primary) immunecomplexes is generally a matter of simply adding the antigen compositionto the sample. One then incubates the mixture for a period of timesufficient to allow the added antigens to form immune complexes with,i.e., to bind to, any antibodies present within the sample. After thistime, the sample composition, such as a tissue section, ELISA plate, dotblot or western blot, will generally be washed to remove anynon-specifically bound antigen species, allowing only those specificallybound species within the immune complexes to be detected.

The detection of immunocomplex formation is well known in the art andmay be achieved through the application of numerous approaches known tothe skilled artisan and described in various publications, such as,e.g., Nakamura et al. (1987), incorporated herein by reference.Detection of primary immune complexes is generally based upon thedetection of a label or marker, such as a radioactive, fluorescent,biological or enzymatic label, with enzyme tags such as alkalinephosphatase, urease, horseradish peroxidase and glucose oxidase beingsuitable. The particular antigen employed may itself be linked to adetectable label, wherein one would then simply detect this label,thereby allowing the amount of bound antigen present in the compositionto be determined.

Alternatively, the primary immune complexes may be detected by means ofa second binding ligand that is linked to a detectable label and thathas binding affinity for the first protein or peptide. The secondbinding ligand is itself often an antibody, which may thus be termed a“secondary” antibody. The primary immune complexes are contacted withthe labeled, secondary binding ligand, or antibody, under conditionseffective and for a period of time sufficient to allow the formation ofsecondary immune complexes. The secondary immune complexes are thengenerally washed to remove any non-specifically bound labeled secondaryantibodies and the remaining bound label is then detected.

For diagnostic purposes, it is proposed that virtually any samplesuspected of containing the antibodies of interest may be employed.Exemplary samples include clinical samples obtained from a patient suchas blood or serum samples, cerebrospinal, synovial, or bronchoalveolarfluid, ear swabs, sputum samples, middle ear fluid or even perhaps urinesamples may be employed. Such methods may be useful for the diagnosisand treatment of various cellular disorders, and in particular, cancersand related conditions.

Furthermore, it is contemplated that such embodiments may haveapplication to nonclinical samples, such as in the titering of antibodysamples, in the selection of hybridomas, and the like. Alternatively,the clinical samples may be from veterinary sources and may include suchdomestic animals as cattle, sheep, and goats. Samples from murine,ovine, opine, caprine, feline, canine, and equine sources may also beused in accordance with the methods described herein.

In related embodiments, the present invention contemplates thepreparation of kits that may be employed to detect the presence ofPS-specific antibodies in a sample. Generally speaking, kits inaccordance with the present invention will include a suitable lipid,lipid/protein or peptide together with an immunodetection reagent, and ameans for containing the lipid, protein or peptide and reagent.

The immunodetection reagent will typically comprise a label associatedwith a PS antigen composition, or associated with a secondary bindingligand. Exemplary ligands might include a secondary antibody or lipidbinding protein directed against the first PS antigen or antibodycomposition, or a biotin or avidin (or streptavidin) ligand having anassociated label. Detectable labels linked to antibodies that havebinding affinity for a human antibody are also contemplated, e.g., forprotocols where the first reagent is a PS antigen composition that isused to bind to a reactive antibody from a human sample. Of course, asnoted above, a number of exemplary labels are known in the art and allsuch labels may be employed in connection with the present invention.The kits may contain antigen, lipid binding protein, or antibody-labelconjugates either in fully conjugated form, in the form ofintermediates, or as separate moieties to be conjugated by the user ofthe kit.

The container means will generally include at least one vial, test tube,flask, bottle, syringe or other container means, into which the antigenmay be placed, and preferably suitably allocated. Where a second bindingligand is provided, the kit will also generally contain a second vial orother container into which this ligand or antibody may be placed. Thekits of the present invention will also typically include a means forcontaining the vials in close confinement for commercial sale, such as,e.g., injection or blow-molded plastic containers into which the desiredvials are retained.

2.3 Immunodetection Kits and Methods

Another aspect of the invention are immunodetection kits containinglipid or lipid-carrier conjugate antigen-specific antibodies andsuitable immunodetection reagents such as a detectable label linked to aprotein, peptide or the antibody itself. Alternatively, the detectablelabel may be linked to a second antibody which binds to a lipid-specificantibody as disclosed herein.

Related embodiments include diagnostic and therapeutic kits whichinclude pharmaceutically-acceptable formulations of either theantibodies, lipid, lipid/peptide, or peptide antigens disclosed herein.Such kits are useful in the detection of lipids such as PS in clinicalsamples, and also useful for promoting an immune response in an animal,and in the formulation of vaccine compositions effective in thetreatment of a variety of cancers.

2.4 Vaccine Formulation and Compositions

In certain embodiments, the inventor contemplates the use of thelipid-carrier conjugate compositions for the preparation of anti-cancervaccines or treatment regimens for administration to an animal, and inparticular, a human. It is expected that to achieve an “immunologicallyeffective formulation” it may be desirable to administer a lipid-carrierconjugate composition, such as a PS-carrier antigen composition, to thehuman or animal subject in a pharmaceutically acceptable compositioncomprising an immunologically effective amount of an antigen compositionmixed with other excipients, carriers, or diluents which may improve orotherwise alter stimulation of B cell and/or T cell responses, orimmunologically inert salts, organic acids and bases, carbohydrates, andthe like, which promote stability of such mixtures. Immunostimulatoryexcipients, often referred to as adjuvants, may include salts ofaluminum (often referred to as Alums), simple or complex fatty acids andsterol compounds, physiologically acceptable oils, polymericcarbohydrates, chemically or genetically modified protein toxins, andvarious particulate or emulsified combinations thereof. Lipid conjugateantigen compositions within these mixtures, or each variant if more thanone are present, would be expected to comprise about 0.0001 to 1.0milligrams, or more preferably about 0.001 to 0.1 milligrams, or evenmore preferably less than 0.1 milligrams per dose.

2.5 Therapeutic and Diagnostic Kits Comprising Lipid-conjugate Antigentsor Lipid-specific Antibody Compositions

A therapeutic kit comprising, in suitable container means, one or morelipid-conjugate antigen(s) or antibody composition(s) of the presentinvention in a pharmaceutically acceptable formulation, representsanother important aspect of the invention.

The kit may comprise a single container means that contains thelipid-conjugate antigen(s) or antibody composition(s). The containermeans may, if desired, contain a pharmaceutically acceptable sterileexcipient, having associated with it, the lipid-conjugate antigen(s) orantibody composition(s) and, optionally, a detectable label or imagingagent. The formulation may be in the form of a gelatinous composition(e.g., a collagenous composition), a powder, solution, matrix,lyophilized reagent, or any other such suitable means. In certain cases,the container means may itself be a syringe, pipette, or other such likeapparatus, from which the lipid-conjugate antigen(s) or antibodycomposition(s) may be applied to a tissue site, tumor, skin lesion,wound area, or other site of administration. However, the singlecontainer means may contain a dry, or lyophilized, mixture of one ormore lipid-conjugate antigen(s) or antibody composition(s), which may ormay not require pre-wetting before use.

Alternatively, the kits of the invention may comprise distinct containermeans for each component. In such cases, one or more containers wouldcontain each of the PS composition(s), either as sterile solutions,powders, lyophilized forms, etc., and the other container(s) wouldinclude a matrix, solution, or other suitable delivery device forapplying the composition to the body, bloodstream, or to a tissue site,skin lesion, tumor cell, wound area, or other site of administration.Such delivery device may or may not itself contain a sterile solution,diluent, gelatinous matrix, carrier or other pharmaceutically-acceptablecomponents.

The kits may also comprise a second or third container means forcontaining a sterile, pharmaceutically acceptable buffer, diluent orsolvent. Such a solution may be required to formulate thelipid-conjugate antigen(s) or antibody composition(s) into a moresuitable form for application to the body, e.g., as a topicalpreparation, or alternatively, in oral, parenteral, or intravenousforms. It should be noted, however, that all components of a kit couldbe supplied in a dry form (lyophilized), which would allow for “wetting”upon contact with body fluids. Thus, the presence of any type ofpharmaceutically acceptable buffer or solvent is not a requirement forthe kits of the invention. The kits may also comprise a second or thirdcontainer means for containing a pharmaceutically acceptable detectableimaging agent or composition.

The container means will generally be a container such as a vial, testtube, flask, bottle, syringe or other container means, into which thecomponents of the kit may placed. The components may also be aliquotedinto smaller containers, should this be desired. The kits of the presentinvention may also include a means for containing the individualcontainers in close confinement for commercial sale, such as, e.g.,injection or blow-molded plastic containers into which the desired vialsor syringes are retained.

Irrespective of the number of containers, the kits of the invention mayalso comprise, or be packaged with, an instrument for assisting with theplacement of the lipid-carrier conjugate, or antibodies reactivetherewith, within the body of an animal. Such an instrument may be asyringe, needle, surgical instrument, pipette, forceps, or any suchmedically approved delivery vehicle.

2.6 Antibody Composition and Formulations Thereof

As described above, an important embodiment of the invention is theformulation of lipid-specific antibodies which are useful in detectingand treating various cancers in an animal, and particularly, in a human.Means for preparing and characterizing antibodies are well known in theart (See, e.g., Harlow and Lane (1988); incorporated herein byreference). The methods for generating mAbs generally begin along thesame lines as those for preparing polyclonal antibodies. Briefly, apolyclonal antibody is prepared by immunizing an animal with one or moreof the lipid-carrier protein compositions disclosed herein andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically the animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster, a guinea pig or a goat. Because of the relatively large bloodvolume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. With respect to preparing lipid-specific antibodies, itis necessary to boost the host immune system, and may be achieved bycoupling the lipid of interest, such as PS, to a carrier. As describedabove, exemplary and preferred carriers include polypeptide carrierssuch as KLH, BSA, and β₂-glycoprotein I. Other albumins such asovalbumin, mouse serum albumin or rabbit serum albumin can also be usedas carriers, as well as bovine gamma globulin and/or diphtheria toxoid.Although means for conjugating lipids to a carrier protein arewell-known in the art, two particular synthesis methods are disclosedherein which have been particularly useful in preparing covalentlipid-specific antibody formulations.

mAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition. The immunizing compositionis administered in a manner effective to stimulate antibody producingcells. Rodents such as mice and rats are preferred animals, however, theuse of rabbit, sheep or frog cells is also possible. The use of rats mayprovide certain advantages (Goding, 1986), but mice are preferred, withthe BALB/c mouse being most preferred as this is most routinely used andgenerally gives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B-lymphocytes (B-cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately about 5×10⁷ to about 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fuision efficiency, and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, 1986; Campbell, 1984). For example, wherethe immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653,NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 andS194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all usefulin connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMistein, 1975; 1976), and those using polyethylene glycol (PEG), such as37% (vol./vol.) PEG, by Gefter et al. (1977). The use of electricallyinduced fusion methods is also appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to about 1×10⁻⁸. However, this does not pose a problem, asthe viable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimnmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fuision. The injected animal develops tumorssecreting the specific mAb produced by the fused cell hybrid. The bodyfluids of the animal, such as serum or ascites fluid, can then be tappedto provide mabs in high concentration. The individual cell lines couldalso be cultured in vitro, where the mAbs are naturally secreted intothe culture medium from which they can be readily obtained in highconcentrations. mAbs produced by either means may be further purified,if desired, using filtration, centrifugation and various chromatographicmethods such as HPLC or affinity chromatography.

3.0 BRIEF DESCRIPTION OF THE DRAWINGS

The drawings form part of the present specification and are included tofurther demonstrate certain aspects of the present invention. Theinvention may be better understood by reference to one or more of thesedrawings in combination with the detailed description of specificembodiments presented herein.

FIG. 1. Synthesis method B for producing PS-carrier antigenicconjugates. SPDP-PS is “deblocked” with (tris[2-carboxyethyl]phosphineHCl) to yield a free sulfhydryl which is then directly coupled tomaleimide activated carrier proteins.

FIG. 2. Synthesis method A for producing of PS-carrier conjugateantigenic conjugates. NH₂-PC was acylated with SPDP, converted to the PSderivative with phospholipase D and coupled by thiol-disulphide exchangeto carrier protein.

FIG. 3A. Reactivity of rabbit PS antiserum with phospholipids.Microtiter plates were coated with 6 μg of lipid. Bound IgG wasquantified by ELISA with peroxidase conjugated goat anti-rabbit Ig.Binding of control pre-immune (∘) and antiserum () to PS coated plates.

FIG. 3B. Reactivity of rabbit PS antiserum with phospholipids.Microtiter plates were coated with 6 μg of lipid. Bound IgG wasquantified by ELISA with peroxidase conjugated goat anti-rabbit Ig.Binding of PS antiserum to polystyrene plates coated with 6 μg of DOPE(▪), PA (▴), PG (▾) and PC (♦).

FIG. 3C. Reactivity of rabbit PS antiserum with phospholipids.Microtiter plates were coated with 6 μg of lipid. Bound IgG wasquantified by ELISA with peroxidase conjugated goat anti-rabbit Ig.Binding of PS antiserum to polystyrene plates coated with PS/PC(1/1)(), DOPE/PC(1/1) (▪).

FIG. 4A. Inhibition of immune serum binding with soluble head groupanalogs and vesicles. The binding assays shown in FIG. 3 were carriedout on PS-coated plates in the presence of glycerophosphoserine (GPS),phosphoserine (PhoS), serine (S), glycerophosphoethanolarnine (GPE),phosphoethanolamine (PhoE), and ethanolamnine (E) at the indicatedconcentrations.

FIG. 4B. Inhibition of immune serum binding with soluble head groupanalogs and vesicles. The binding assays shown in FIG. 3 were carriedout on DOPE-coated plates in the presence of GPE and PhoE at theindicated concentrations.

FIG. 4C. Inhibition of immune serum binding with soluble head groupanalogs and vesicles. Plates coated with PS and DOPE were assessed forantibody binding in the presence of sonicated vesicles (0.5 mg/ml)containing PS (PS/PC,1/1), DOPE (PE/PC,1/1) and PC.

FIG. 5. Inhibition of prothrombinase activity. Prothrombinase activitywas assessed in the presence of PS-containing vesicles preincubated withthe indicated sera. The reaction was stopped at various time points withEDTA and thrombin production was assessed by determining the initialrates of thrombin-dependent cleavage of the chromogenic substrate. Theserates were plotted on the ordinate against the incubation time.Pre-immune serum (∘), antiserum ().

FIG. 6A. Fluorescence microscopy and analysis by flow cytometry ofanti-PS treated red blood cells (RBC). Papain-treated RBC were incubatedfor 1 h with A23187 (5 μM) and Ca²⁺ (1 mM) followed by labeling with theantiserum and fluorescein-conjugated anti-rabbit IgG. Phasephotoricrographs of antibody-labeled PS-expressing RBC.

FIG. 6B. Fluorescence microscopy and analysis by flow cytometry ofanti-PS treated red blood cells. Papain-treated RBC were incubated for 1h with A23187 (5 μM and Ca²⁺ (1 mM) followed by labeling with theantiserum and fluorescein-conjugated anti-rabbit IgG. Fluorescencephotomicrographs of antibody-labeled PSxpressing RBC.

FIG. 6C. Fluorescence microscopy and analysis by flow cytometry ofanti-PS treated RBC. Papain-treated RBC were incubated for 1 h withA23187 (5 μM) and Ca²⁺ (1 mM) followed by labeling with the controlserum and fluorescein-conjugated anti-rabbit IgG. Flow cytometryanalysis of PS-expressing RBC incubated with control sera.

FIG. 6D. Fluorescence microscopy and analysis by flow cytometry ofanti-PS treated RBC. Papain-treated RBC were incubated for 1 h withA23187 (5 μM) and Ca²⁺ (1 mM) followed by labeling with the antiserumand fluorescein-conjugated anti-rabbit IgG. Flow cytometry analysis ofPS-expressing RBC incubated with immune sera.

FIG. 7. C57B1/6 mice were inoculated subcutaneously (s.c.) withPS-expressing (determined by the ability of the cells to be stained withthe PS-specific reagent, fluoresceinconjugated annexin V) EG7 lymphomacells. The mice were sorted into treatment groups (6-8 animals/group)when tumors ranged in size between approximately 75-100 mm³ at whichtime immunization was initiated. The tumor-bearing mice were immunizedwith a single injection of Provax on day 8 with 100 μg of PS-KLH orPS-BSA conjugate. Tumor growth delays ranged from 38 days for the PS-BSAgroup and 20 days for the PS-KLH group.

4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS 4.1 Affinity Chromatography

Affinity chromatography is generally based on the recognition of aprotein by a substance such as a ligand or an antibody. The columnmaterial may be synthesized by covalently coupling a binding molecule,such as an activated dye, for example to an insoluble matrix. The columnmaterial is then allowed to adsorb the desired substance from solution.Next, the conditions are changed to those under which binding does notoccur and the substrate is eluted. The requirements for successfulaffinity chromatography are:

1) that the matrix must specifically-adsorb the molecules of interest;

2) that other contaminants remain unadsorbed;

3) that the ligand must be coupled without altering its bindingactivity;

4) that the ligand must bind sufficiently tight to the matrix; and

5) that it must be possible to elute the molecules of interest withoutdestroying them.

A preferred embodiment of the present invention is an affinitychromatography method for purification of antibodies from solutionwherein the matrix contains a lipid such as PS, or a lipid-conjugateantigen composition, such as a PS-protein conjugate, covalently-coupledto a matrix such as Sepharose CL6B or CL4B. Such a matrix binds thePS-specific antibodies of the present invention directly and allowstheir separation by elution with an appropriate gradient such as salt,GuHCl, pH, or urea. Another preferred embodiment of the presentinvention is an affinity chromatography method for the purification oflipid-conjugate antigen compositions from solution In such methods, thematrix would comprise antibodies which specifically bind to thelipid-onjugate antigen compositions of the present invention directly,thus permitting their separation by elution with a suitable buffer asdescribed above.

4.2 Liposomes and Nanocapsules

In certain embodiments, the inventor contemplates the use of liposomesand/or nanocapsules for the introduction of particular antigens orantibodies into host cells. Such formulations may be preferred for theintroduction of pharmaceutically-acceptable formulations of thelipid-carrier polypeptide conjugates and/or antibodies disclosed herein.The formation and use of liposomes is generally known to those of skillin the art (see for example, Couvreur et al., 1977 which describes theuse of liposomes and nanocapsules in the targeted antibiotic therapy ofintracellular bacterial infections and diseases). More recently,liposomes were developed with improved serum stability and circulationhalf-times (Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987).

In a further embodiment of the invention, the PS/potypeptide compositionmay be entrapped in a fiposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991).

Nanocapsules can generally entrap compounds in a stable and reproducibleway (Henry-Michelland et al., 1987). To avoid side effects due tointracellular polymeric overloading, such ultrafine particles (sizedaround 0.1 μm) should be designed using polymers able to be degraded invivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meetthese requirements are contemplated for use in the present invention,and such particles may be are easily made, as described (Couvreur etal., 1977; 1988).

4.3 Methods for Preparing Lipid-specific Antibodies

In another aspect, the present invention contemplates an antibody thatis immunoreactive with a lipid such as PS. As stated above, one of theuses for lipid-carrier conjugate antigen compositions according to thepresent invention is to generate antibodies. Reference to antibodiesthroughout the specification includes whole polyclonal and monoclonalantibodies (mAbs), and parts thereof, either alone or conjugated withother moieties. Antibody parts include Fab and F(ab)₂ fragments andsingle chain antibodies. The antibodies may be made in vivo in suitablelaboratory animals or in vitro using recombinant DNA techniques. In apreferred embodiment, an antibody is a polyclonal antibody. .Briefly, apolyclonal antibody is prepared by immunizing an aimgal with animmunogen comprising a lipid/polypeptide of the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically an animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster or a guinea pig. Because of the relatively large blood volume ofrabbits, a rabbit is a preferred choice for production of polyclonalantibodies.

Antibodies, both polyclonal and monoclonal, specific for lipids such asPS may be prepared using conventional immunization techniques, as willbe generally known to those of skill in the art. A compositioncontaining lipid antigen compositions described herein may be used toimmunize one or more experimental animals, such as a rabbit or mouse,which will then proceed to produce specific antibodies against lipidssuch as PS. Polyclonal antisera may be obtained, after allowing time forantibody generation, simply by bleeding the animal and preparing serumsamples from the whole blood.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen, as well as theanimal used for immunization. A variety of routes can be used toadminister the immunogen (subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal). The production of polyclonalantibodies may be monitored by sampling blood of the immunized animal atvarious points following immunization. A second, booster injection, alsomay be given. The process of boosting and titering is repeated until asuitable titer is achieved. When a desired level of immunogenicity isobtained, the immunized animal can be bled and the serum isolated andstored, and/or the animal can be used to generate mAbs (below).

One of the important features provided by the present invention is apolyclonal sera that is relatively homogenous with respect to thespecificity of the antibodies therein. Typically, polyclonal antisera isderived from a variety of different “clones,” i.e., B-cells of differentlineage. mAbs, by contrast, are defined as coming fromantibody-producing cells with a common B-cell ancestor, hence their“mono” clonality.

To obtain mAbs, one would also initially immunize an experimentalanimal, often preferably a mouse, with a lipid-carrier proteinconjugate-containing composition. One would then, after a period of timesufficient to allow antibody generation, obtain a population of spleenor lymph cells from the animal. The spleen or lymph cells can then befused with cell lines, such as human or mouse myeloma strains, toproduce antibody-secreting hybridomas. These hybridomas may be isolatedto obtain individual clones which can then be screened for production ofantibody to the desired peptide.

Following immunization, spleen cells are removed and fused, using astandard fuision protocol with plasmacytoma cells to produce hybridomassecreting mAbs against the antigen compositions. Hybridomas whichproduce mAbs to the selected antigens are identified using standardtechniques, such as ELISA and Western blot methods. Hybridoma clones canthen be cultured in liquid media and the culture supernatants purifiedto provide the lipid-specific mAbs.

It is proposed that the mAbs of the present invention will also finduseful application in immunochemical procedures, such as ELISA andWestern blot methods, as well as other procedures such asimmunoprecipitation, immnunocytological methods, etc. which may utilizeantibodies specific to lipids such as PS. In particular, lipid-specificantibodies may be used in immunoabsorbent or affinity protocols asdescribed above to purify lipid-containing compositions. The operationof all such immunological techniques will be known to those of skill inthe art in light of the present disclosure.

4.4 Immunoassays

As noted, it is proposed that the lipid-carrier conjugate compositionsof the invention will find utility as immunogens, e.g., in connectionwith vaccine development, or as antigens in immunoassays for thedetection of reactive antibodies. Turning first to immunoassays, intheir most simple and direct sense, preferred immunoassays of theinvention include the various types of enzyme linked immunosorbentassays (ELISAs), as are known to those of skill in the art. However, itwill be readily appreciated that the utility of lipid-carrier conjugatecompositions is not limited to such assays, and that other usefulembodiments include RIAs and other non-enzyme linked antibody bindingassays aid procedures.

In preferred ELISA assays, proteins or peptides incorporatinglipid-carrier conjugate compositions are immobilized onto a selectedsurface, preferably a surface exhibiting a protein affinity, such as thewells of a polystyrene microtiter plate. After washing to removeincompletely adsorbed material, one would then generally desire to bindor coat a nonspecific protein that is known to be antigenically neutralwith regard to the test antisera, such as bovine serum albumin (BSA) orcasein, onto the well. This allows for blocking of nonspecificadsorption sites on the immobilizing surface and thus reduces thebackground caused by nonspecific binding of antisera onto the surface.

After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with theantisera or clinical or biological extract to be tested in a mannerconducive to immune complex (antigen/antibody) formation. Suchconditions preferably include diluting the antisera with diluents suchas BSA, bovine gamma globulin (BGG) and phosphate buffered saline(PBS)/Tween®. These added agents also tend to assist in the reduction ofnonspecific background. The layered antisera is then allowed to incubatefor, e.g., from 2 to 4 hours, at temperatures preferably on the order ofabout 25° to about 27° C. Following incubation, the antisera-contactedsurface is washed so as to remove non-immunocomplexed material. Apreferred washing procedure includes washing with a solution such asPBSITween®, or borate buffer.

Following formation of specific immunocomplexes between the test sampleand the bound lipid-carrier conjugate composition, and subsequentwashing, the occurrence and the amount of immunocomplex formation may bedetermined by subjecting the complex to a second antibody havingspecificity for the first. Of course, in that the test sample willtypically be of human origin, the second antibody will preferably be anantibody having specificity for human antibodies. To provide a detectingmeans, the second antibody will preferably have an associated detectablelabel, such as an enzyme label, that will generate a signal, such ascolor development upon incubating with an appropriate chromogenicsubstrate. Thus, for example, one will desire to contact and incubatethe antisera-bound surface with a urease or peroxidase-conjugatedanti-human IgG for a period of time and under conditions that favor thedevelopment of immunocomplex formation (e.g., incubation for 2 hours atroom temperature in a PBS-containing solution such as PBS-Tween®)).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline)sulfonic acid (ABTS) andH₂O₂, in the case of peroxidase as the enzyme label. Quantitation isthen achieved by measuring the degree of color generation, e.g., using avisible spectrum spectrophotometer.

ELISAs may be used in conjunction with the invention. In one such ELISAassay, proteins or peptides incorporating antigenic sequences ormoieties of the present invention are immobilized onto a selectedsurface, preferably a surface exhibiting a protein affinity such as thewells of a polystyrene microtiter plate. After washing to removeincompletely adsorbed material, it is desirable to bind or coat theassay plate wells with a nonspecific protein that is known to beantigenically neutral with regard to the test antisera such as bovineserum albumin (BSA), casein or solutions of powdered milk. This allowsfor blocking of nonspecific adsorption sites on the immobilizing surfaceand thus reduces the background caused by nonspecific binding ofantisera onto the surface.

4.5 Immunoprecipitation

The lipid-carrier conjugate-specific antibodies of the present inventionare particularly useful for the isolation of lipid-containingcompositions by immunoprecipitation. Immunoprecipitation involves theseparation of the target antigen component from a complex mixture, andis used to discriminate or isolate minute amounts of protein. For theisolation of cell-surface localized compositions, such as PS, thesecompositions may be solubilized from the cell by treatment with enzymes,or alternatively, into detergent micelles. Nonionic salts are preferred,since other agents such as bile salts, precipitate at acid pH or in thepresence of bivalent cations.

In an alternative embodiment the antibodies of the present invention areuseful for the close juxtaposition of two antigens. This is particularlyuseful for increasing the localized concentration of antigens, e.g.,enzyme-substrate pairs.

4.6 Western Blots

The lipid antigen and lipid-specific antibody compositions of thepresent invention find great use in a variety of immunoblot and westernblot analyses. For example, the PS-specific antibodies may be used ashigh-affinity primary reagents for the identification of PS-containingcompositions immobilized onto a solid support matrix, such asnitrocellulose, nylon or combinations thereof. In conjunction withimmunoprecipitation, followed by gel electrophoresis, these may be usedas a single step reagent for use in detecting antigens against whichsecondary reagents used in the detection of the antigen cause an adversebackground. Immunologically-based detection methods in conjunction withWestern blotting (including enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the toxin moiety) areconsidered to be of particular use in this regard.

4.7 PS Compositions for Treating Cancer

The maintenance of a particular lipid bilayer equilibrium distribution,in particular the preservation of PS in the cell's inner leaflet, is aproperty characteristic of normal, mature cells. If the translocationmachinery becomes impaired, such as in tumor cells, PS appears at thecell surface and invokes substantial functional consequences.

The inventor contemplates that the PS compositions described herein maybe used for the prevention of or the treatment of essentially anydisorder that is characterized by the presence of PS on the surface ofthe cell. Cancers having such a characteristic may include those of thebrain, lung, liver, spleen, kidney, bladder, lymph node, smallintestine, pancreas, blood cells, colon, stomach, breast, endometrium,prostate, testicle, ovary, skin, head and neck, esophagus, or bonemarrow. In preferred embodiments, the cancerous cells are derived fromthe kidney, bladder, lymph nodes or bone marrow.

4.8 Pharmaceatical Compositions

In certain embodiments, the pharmaceutical compositions disclosed hereinmay be orally administered, for example, with an inert diluent or withan assimilable edible carrier, or they may be enclosed in hard or softshell gelatin capsule, or they may be compressed into tablets, or theymay be incorporated directly with the food of the diet. For oraltherapeutic administration, the active compounds may be incorporatedwith excipients and used in the form of ingestible tablets, buccaltables, troches, capsules, elixirs, suspensions, syrups, wafers, and thelike. Such compositions and preparations should contain at least 0.1% ofactive compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 2 toabout 60% of the weight of the unit. The amount of active compounds insuch therapeutically useful compositions is such that a suitable dosagewill be obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, as gum tragacanth, acacia, cornstarch, or gelatin;excipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; and a sweetening agent, such as sucrose, lactoseor saccharin may be added or a flavoring agent, such as peppermint, oilof wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and propylparabens as preservatives, a dye and flavoring,such as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

Alternatively, in some embodiments, it may be desirable to administerthe antigen or antibody compositions disclosed either intravenously,parenterally or intraperitoneally. Solutions of the active compounds asfree base or pharmacologically acceptable salts can be prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it win be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuumdrying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

For oral prophylaxis the polypeptide may be incorporated with excipientsand used in the form of non-ingestible mouthwashes and dentifrices. Amouthwash may be prepared incorporating the active ingredient in therequired amount in an appropriate solvent, such as a sodium boratesolution (Dobell's Solution). Alternatively, the active ingredient maybe incorporated into an antiseptic wash containing sodium borate,glycerin and potassium bicarbonate. The active ingredient may also bedispersed in dentifrices, including: gels, pastes, powders and slurries.The active ingredient may be added in a therapeutically effective amountto a paste dentifrice that may include water, binders, abrasives,flavoring agents, foaming agents, and humectants.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

The composition can be formulated in a neutral or salt form.Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug release capsules and the like.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

4.9 Vaccine Preparation

The compositions described herein provide immunogenic particles that areable to elicit an anti-PS immune response. Because PS generally is foundon the surface of aberrant cell types (e.g., tumor cells and apoptoticcells), the inventor contemplates that such compositions are ideal foruse as a potential vaccine against tumorigenesis. Thus the presentinvention provides an immunogenic composition that may be used as avaccine against cancer.

In certain embodiments, such vaccines may be injectable liquid solutionsor emulsions. The PS compositions disclosed herein may be mixed withpharmaceutically-acceptable excipients which are compatible with the PScompositions. By compatible it is meant that thephamaceutically-acceptable excipients will not alter the conformationalcharacteristics of the immunogen. Excipients may include water, saline,dextrose, glycerol, ethanol, or combinations thereof. The vaccine mayfurther contain auxiliary substances, such as wetting or emulsifyingagents, buffering agents, or adjuvants to enhance the effectiveness ofthe vaccines. Adjuvants may be mineral salts (e.g., AlK(SO₄)₂,AlNa(SO₄)₂, AlNH₄(SO₄), silica, alum, Al(OH)₃, Ca₃(PO₄), kaolin, orcarbon), polynucleotides (e.g., poly IC or poly AU acids), and certainnatural substances (e.g., wax D from Mycobacterium tuberculosis,substances found in Corynebacterium parvum, Bordetella pertussis, ormembers of the genus Brucella)(Int. Pat. Appl. Publ. No. WO 91/09603).Aluminum hydroxide or phosphate (alum) are commonly used at 0.05 to 0.1percent solution in phosphate buffered saline. Other adjuvant compoundsinclude QS2 or incomplete Freunds adjuvant. A preferred adjuvant isProvax (IDEC Pharmaceuticals).

Vaccines may be administered parenterally, by injection subcutaneouslyor intramuscularly, or the vaccines may be formulated and delivered toevoke an immune response at the mucosal surfaces. The immunogeniccomposition may be administered to a mucosal surface by the nasal, oral,vaginal, or anal routes. For anal delivery, suppositories may be used.Suppositories may comprise binders and carriers such as polyalkaleneglycols or triglycerides. Oral formulations may be in the form of pills,capsules, suspensions, tablets, or powders and include pharmaceuticalgrades of saccharine, cellulose or magnesium carbonate. Thesecompositions may contain from about 5% to about 95% of the PScomposition or more as needed.

Preferably the vaccines are administered in a manner and amount as to betherapeutically effective. That is to say that the vaccine should beadministered in such a way as to elicit an immune response to PS.Suitable doses required to be administered are readily discernible bythose of skill in the art. Suitable methodologies for the initialadministration and booster doses, if necessary, maybe variable also. Thedosage of the vaccine may depend on the route of administration and mayvary according to the size of the host.

Although the immunogenic compositions of the present invention may beadministered to individuals that have not been diagnosed with cancer,they also may be administered to individuals who have been diagnosedwith cancer in an effort to alter the immune response to the tumor. Thealteration may be an increase in antibody production, a stimulation ofanti-tumor CD4⁺ or CD8⁺ T cells, or in respect to the type of responseto the virus (i.e., T_(H)1 vs. T_(H)2). Nonetheless, this alteration, ifeffective, will decrease the mortality and morbidity associated with thecancer. In other words, the immunogenic compound may decrease theseverity of the disease and increase the life of the patient.

4.10 Contemporary Cancer Therapy

Most attempts to promote a therapeutic immune response against cancerhave been directed towards unique, tumor-specific, peptide orcarbohydrate antigens. Little or no attention, however, has been givento the possibility that specific anti-lipid responses might also beexploited for this purpose. Although phospholipids are ubiquitous, it isclear that the organization and membrane sidedness of individual lipidspecies is not random but is controlled by transport mechanisms thatmaintain specific transmembrane lipid distributions (Devaux andZachowski, 1994; Menon, 1995). Recent data suggests that while membraneorganization is tightly regulated over the lifespan of the cell, normallipid distributions are not maintained upon the cell's acquisition ofseveral pathologic phenotypes (Zwaal and Schroit, 1997). This isparticularly evident for tumorigenic cells where phosphatidylserine (PS)redistributes from the cell's inner leaflet (its normal location) to theouter leaflet upon transformation (Connor et al., 1989; Utsugi et al.,1991). This condition raises the possibility that PS on the cell's outerleaflet can serve as a target for therapeutic intervention.

4.11 Membrane Lipid Asymmetry and Recognition of PS-expressing Cells

The outer leaflet of eukaryotic cell membranes contains most of thecholinephospholipids, whereas the aminophospholipids are mainly presentin the cell's inner leaflet (Verkleij et al., 1973; Zwaal et al., 1975).Although asymmetry seems to be the rule for normal cells, loss ofmembrane lipid sidedness, in particular the emergence of PS at the cellsurface, results in the expression of altered surface properties thatmodulates cell function and influences the cell's interaction with itsenvironment (Zwaal and Schroit, 1997). For example, the exposure of PSpromotes coagulation and thrombosis by platelets (Bevers et al., 1982)and is involved in the recognition and elimination of apoptotic cells(Fadok et al., 1992), senescent cells (Connor et al., 1994) andtumorigenic cells (Utsugi et al., 1991) by phagocytes.

4.12 Autoimmunity and Cancer

Antiphospholipid antibodies (APA) have been demonstrated mainly in seraof patients with connective tissue disease, particularly systemic lupuserythematosus (Mackworth-Young, 1990; Asherson and Cervera, 1993).Although less frequent, APA have also been detected in patients withmalignancies, including leukemia, lymphoma, epithelial malignancies andthymoma (Becker and Brocker, 1995; Naldi et al., 1992). Recent studiesshowed that APA levels were significantly higher in melanoma patientswho received immunotherapy with interferon-α or bacillus Calmette-Guerin(Becker et al., 1994; Herstoff and Bogaars, 1979). Furthermore, ongoingpreclinical studies investigating the relationship between autoimmunityin leukemia patients treated with interferon-α showed a strongrelationship between hematologic/clinical remissions and the levels ofAPA. Because autoantibodies in patients with autoimmune diseases arecapable of binding and killing cells that display the autoantigens, itis possible that the appearance of APA in some cancers, possibly as aconsequence of the disease and/or treatment regimen, is responsible forthe remissions commonly seen upon interferon-α treatment.

Because PS seems to be a ubiquitous marker for cancer cells, it mayserve as a specific epitope for tumor cell populations and a therapeutictarget for cancer treatment. To test the feasibility of this approach,an autoimmune APA syndrome-like response against PS was raised in miceusing unique immunogens that preserve the lipid's critical head-groupand presents PS as a carrier-bound hapten. Studies have shown that miceimmunized with these PS-carrier systems using Provax adjuvant (IDECPharmaceuticals) are protective against the growth of several syngeneiccarcinomas.

5.0 EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

5.1 Example 1 Production and Characterization of Polyclonal PSAntibodies

Because PS can be considered to be a non-immunogenic hapten, theinventor reasoned that an appropriate lipid-protein conjugate mightelicit a potent and specific immune response. To address this issue andovercome the inherent problems of lipid immunogenicity andcross-reactivity, the inventor synthesized PS that contained a“sulfhydryl-activated” coupling group at the end of the 2-position sidechain and covalently linked the lipid to a protein carrier (Diaz et al.,1998). The inventor shows that this antigen induced the production ofPS-specific antibodies in primates, rabbits and mice and that theseantibodies bound specifically to PS-expressing red blood cells (RBC).The inventor's results suggest that PS antibodies could be an importanttool for the study of PS-dependent processes and its distribution in themembranes of living cells.

5.1.1 Materials and Methods

5.1.1.1 Materials

PS, dioleoylphosphatidic acid (PA), PC, dioleoylphosphatidylglycerol(PG), DOPE was purchased from Avanti Biochemicals (Pelham, Ala.).1-acyl-2-(aminocaproyl)phosphatidylcholine (NH₂-PC) was synthesized aspreviously described (Schroit and Madsen, 1983).N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) and 2-iminothiolanewere purchased from Pierce (Rockford, Ill.). Bovine serum albumin (BSA),keyhole limpet hemocyanin (KLH), prothrombin, factor X and analyticalreagents were from Sigma (St. Louis, Mo.). S2238 was purchased from KabiLaboratories (Franklin, Ohio). Human RBC were obtained from healthyvolunteers by venipuncture into heparinized syringes.

5.1.1.2 Synthesis of 1-Acyl-2-N-succinimidyl-3-(2-pyridyldithio)Propionyl(Aminocaproyl)-PS (SPDP-PS)

SPDP-PS was made from SPDP-PC by phospholipase D catalyzed base-exchangein the presence of L-serine (Conifuirius et al., 1990). Briefly, SPDP-PCwas synthesized first by reacting 20 μmol of NH₂-PC [prepared bydeblocking 1-acyl-2-tBOC-aminocaproyl-PC (Schroit and Madsen, 1983)]with 40 μmol of SPDP in 3 ml of CHCl₃/MEOH/triethylamine (1/2/0.015)overnight. CHCl₃ (1 ml) and water (1.8 ml) was added and the lowerorganic phase was removed. Analysis of the product, SPDP-PC, bythin-layer chromatography (TLC) (CHCl₃/MEOH/H₂O; 65/25/4; Rf=−0.4)revealed a single phosphate positive, ninhydrin negative spot. The lipidwas then dried and resuspended in 1 ml of 50% L-serine in 0.1M acetatebuffer, pH 5.6 containing 0.1M CaCl₂. 1 ml of ether and 25 units (70 μl)of phospholipase D was added and the suspension was mixed at 45° C. for3 h and stopped by the addition of EDTA (to 0.2 M). The ether was thenevaporated and the product was resuspended in CHCl₃/MEOH/H₂O (1/2/0.8).Excess L-serine was removed by centrifugation. The product was recoveredfrom the organic phase after the addition of 1 part CHCl₃ and 1 partwater. The organic phase was taken to dryness, dissolved in CHCl₃, andapplied to a 2×30 cm column of activated prewashed silica gel. Thecolumn was washed with 100 ml of CHCl₃, followed by 100 ml aliquots ofCHCl₃ containing increasing MEOH. Analysis of the product which elutedwith CHCL₃/MEOH (6/4) by TLC revealed a single phosphate andninhydrin-positive spot (Rf=−0.2). The purified product was stored inCHCl₃. Electrospray mass spectra analysis calculated for SPDP-PS[C₃₈H₆₃N₃O₁₁PS₂] (M) 833.02, found 832.

5.1.1.3 Coupling of SPDP-PS to Protein Carriers

SPDP-PS was coupled to BSA or KLH after introducing additionalsulthydryls into the proteins with 2-iminothiolane. Briefly, the carrierproteins were solubilized at 10 mg/ml in 10 mM Tris buffer pH 8.0 whichcontained 0.1 mM EDTA. A 100-fold mole excess of 2-iminothiolane wasadded and the reaction was allowed to proceed for 1 h (Jue et al.,1978). The solution was then dialyzed overnight. To ensure theavailability of maximum free sulfhydtyls for coupling, the protein wasreduced with 5 mM dithiothreitol (DTT). DTT was removed immediatelybefore coupling by exclusion chromatography on a Biogel P6 column. Peakfractions were collected, and available sulfhydryls were estimated withEliman's reagent (DTNB) (Riddles et al., 1983). The reduced protein wasthen immediately mixed with 1 mol equivalent of SPDP-PS in {fraction(1/10)}th volume of ETOH. The efficiency of derivatization was estimatedby measuring the release of 2-thiopyridine at 343 nm (Grasseti andMurray, 1967).

5.1.1.4 Immunization Protocol

Rabbits were injected in multiple intradermal sites with ˜1 mg of thelipid-protein conjugates in complete Freund's adjuvant followed by aboost one month later in incomplete Freund's adjuvant. The rabbits werebled two weeks after the last injection.

5.1.1.5 Enzyme-linked Immunoabsorbent Assays (ELISA)

Polystyrene microtitre plates were coated overnight at room temperaturewith 30 μl/well of 200 μg/ml solution of different phospholipids(AVANTI) in CHCl₃/MEOH (1/50). Blocking of the dried plates was carriedout with 10% goat serum in 0.8% NaCl/20 mM Tris, pH 8.0 for 1 h at roomtemperature. Antiserum samples prepared in blocking solution wereapplied to the wells at different dilutions (reported in the figures)for 2 h and binding was assessed by adding anti-rabbit (whole molecule)peroxidase conjugate (SIGMA) at a 1:10,000 dilution in the same bufferfor 2 h. TMB-ELISA (3,3′5,5′ tetramethylbenzidine base, GIBCO BRL) wasused as the substrate. Inhibition of immune serum binding to PS wasdetermined with the head group analogs glycerophosphorylserine (GPS),phosphorylserine (PhoS), serine, glycerophosphorylethanolamine (GPE),phosphorylethanolamine (PhoE), and ethanolamine at 600 μ/ml, 150 μg/ml,and 40 μg/ml. Inhibition with sonicated vesicles (0.5 mg/ml) composed ofPS (50 mol % in PC), DOPE (50 mol % in PC), and PC was achieved by theaddition of equal volumes of liposomes to antiserum samples diluted in10% goat serum. After 1 h of incubation at 37° C., ELISA was carried outas described above.

5.1.1.6 Protherombinase Activity Assay

The PS-dependent prothrombinase assay was carried out as describedpreviously (Diaz et al., 1996) except that sonicated PS vesicles (SUV)were used as the procoagulant surface. Briefly, 0.05 ml of PS SUV (1mg/ml) were incubated with 0.05 ml of antiserum or control preimmuneserum for 15 min at 37° C. The suspension was then added to 0.2 ml ofprothrombinase assay buffer containing Ca²⁺ and the necessarycoagulation factors for the period of time indicated. Aliquots of thesuspension were then transferred to a cuvette containing 1 ml of EDTAbuffer to stop the production of thrombin. The thrombin-dependentchromogen, S2238, was added to the cuvettes (to 0.2 mmol/l), and therate of chromophore formation was monitored at 405 nm with a GilfordResponse Spectrophotometer employing appropriate kinetic software. Theinitial rate of thrombin-dependent chromophore production was determinedfrom the slope of the absorbance curve. These rates were plotted on theordinate against the incubation time.

5.1.1.7 Immunocytochemistry

Ca²⁺-induced scrambling of RBC lipids was done by incubatingpapain-treated RBC (0.25 mg/ml papain, 1 mM EDTA, 2 mM cysteine-HCl inphosphate-buffered saline for 1 h at 37° C.) with 5 μM A23187 in 1 mMCaCl₂ for 1 h at 37° C. After removing red cell vesicles bycentrifugation, the cells were incubated with anti-PS for 1 h at 0° C.The cells were then washed and stained with fluorescein-conjugated goatanti-rabbit IgG.

5.1.1.8 Flow Cytometry

Data acquisition and analysis were done on a Coulter Epics Profile flowcytometer using EPICS elite software. Forward and side angle lightscatter were set to eliminate red cell ghosts. Fluorescence channelswere set logarithinetically.

5.1.2 Results

5.1.2.1 Synthesis Method a for Preparation of PS-carrier Conjugates

To preserve the integrity of the lipids reactive serine headgroup, theinventor generated a carboxyl- and amine-independent reactive disulfidegroup at the acyl side chain, which was done by acylating NH₂—PC withSPDP. The product, containing a protected disulfide β chain, was thenconverted to the PS derivative with phospholipase D (FIG. 2).Lipid/protein coupling was accomplished by disulfide exchange of thelipid haptens to proteins treated with 2-iminothiolane (1 molSPDP-PS/mol SE). Coupling was stoichiometric as estimated by monitoringthe release of 2-thiopyridine. Coupling ratios were typically 20/1 and135/1 for BSA and KLH, respectively (Table 1).

TABLE 1 COUPLING OF SPDP-PS To AVAILABLE SULFHYDRYL-REACTIVE SITES ONCARRIER PROTEINS BSA BSA + Traut's KLH KLH + Traut's SH^(a) 3 20.6 52.9135.8 1-TP release^(b) 100.7% 99.6% ^(a)Reduced sulfhydryls werequantified with DTNB before and after treating the proteins with anexcess of 2-iminothiolane/DTT. The reduced protein was then immediatelymixed with SPDP-PS (mol/mol). ^(b)Coupling efficiency was estimated bycomparing free sulfhydryls on the carrier protein to the release of2-thiopyridine upon the addition of SPDP-PS.

5.1.2.2 Specificity of PS Antibodies

Antisera obtained from rabbits immunized against PS-BSA were tested byELISA for their ability to bind different phospholipids. The data shownin FIG. 3A, FIG. 3B, and FIG. 3C indicate that the antiserum reactedwith PS and DOPE, but not with PC or other negatively chargedphospholipids. To determine whether the reaction to DOPE was polar-headgroup-specific or due to interactions with other structures that mightbe adopted by DOPE (Rauch et al., 1986; Rauch and Janoff, 1990), bindingwas tested against lipids deposited as 50 mol % mixtures with PC. FIG.3C shows that while reactivity to PS was preserved, antibody binding to50 mol % mixtures of DOPE in PC was similar to the levels obtained withPC alone.

To determine which epitope was responsible for PS binding, thereactivity of the antibodies to the lipid's polar head group wasassessed by competitive inhibition with lipid analogs and liposomes ofdifferent lipid composition (FIG. 4A, FIG. 4B, and FIG. 4C). At thehighest concentration tested, GPS and PhoS, inhibited binding to the PScoated plates by ^(˜)80% and 60%, respectively. Serine, GPE, PhoE andethanolamine were without effect (FIG. 4A). Consistent with the resultspresented in FIG. 3C, GPE and PhoE did not inhibit antibody binding toDOPE (FIG. 4B), suggesting that antibody reactivity to DOPE wasindependent of the lipid's polar head group. This was verified by theability of SUV, irrespective of lipid composition, to inhibit binding toDOPE- but not to PS-coated plates (FIG. 4C).

Because these antibodies bind PS, they should also be able to interferewith PS-dependent processes such as coagulation. To test this, PSvesicles were preincubated with the antiserum for 1 h at 37° C. and theability of the vesicles to promote the PS-dependent prothrombinasereaction was assessed. The results presented in FIG. 5 show that theinitial rates of thrombin-dependent S2238 cleavage after the indicatedreaction times were inhibited by ^(˜)60% in the antiserum-treatedsamples (calculated from the slopes of the fitted curve).

5.1.2.3 Generation of PS Antibodies In Primates

Cynomolgus monkeys are immunized with 100 to 250 pg of PS-KLH every twoto three weeks. The monkeys were bled every two weeks and the sera wastested in serial dilutions for anti-PS activity in a direct PS-ELISA.One monkey responded with a reciprocal anti-PS titer of ˜2700. Anti-PStiters slowly declined over a period of 5 months. The PS-BSA conjugate(250 μg) was then used in subsequent immunizations. Reciprocal anti-PStiters steadily increased in both rabbits after each immunization to˜24,300.

5.1.2.4 Detection of Cell Surface PS By Immunofluorescence

Red cells were induced to express PS at the cell surface by Ca⁺² influxwhich results in interleaflet lipid mixing (Sims et al., 1989; Baldwinet al., 1990; Bevers et al., 1992; Gaffet et al., 1995). The presence ofPS on the ionophore treated cells was confirmed by assessing theirPS-dependent prothrombinase activity. Cells treated with ionophore andCa²⁺ were incubated with PS antibodies followed by fluoresceinconjugated anti-Rabbit IgG. Fluorescence microscopy showed that thesecells were strongly fluorescent (FIG. 6B). Cells treated with preimmuneserum were not fluorescent (FIG. 6A) nor were control RBC (cells treatedwith ionophore alone or Ca⁺² alone) incubated with preimmune or immuneserum. Staining of the Ca⁺²/ionophore-treated RBC was also quantified byflow cytometry. Analysis of RBC incubated with antiserum followed byfluorescein-conjugated anti-rabbit IgG showed that 44% of the populationwas within the gated area (FIG. 6D) above the background fluorescence ofcontrol cefls (FIG. 6C).

5.1.3 Discussion

Various methods have been used to determine the presence of PS on cellmembranes. These include direct chemical modification with membraneimpermeable reagents such as trinitrobenzenesulfonic acid and hydrolysiswith specific phospholipases (Gordesky et al., 1975; Etemadi 1980),direct labeling with PS binding proteins (Thiagarajan and Tait, 1990;Tait and Gibson, 1994; Vermes et al., 1995; Kuypers et al., 1996), andPS-dependent catalysis of coagulation (Rosing et al., 1980; Van Dieijenet al, 1981). Several laboratories used lipid antibodies to detect cellsurface PS (Maneta-Peyret et al., 1993; Rote et al., 1993; Rote et al.,1995; Katsuragawa et al, 1995). However, many of these antibodies arenot specific and cross-reactivity is common. This may be due to the weakantigenic presentation of the phosphorylated head groups that arecritical to specificity or to the generation of antibodies todiacylglycerol, phosphodiester and/or fatty acid moieties that arecommon to all phospholipids. In an attempt to produce specific PSantibodies, the inventor immunized rabbits with PS covalently coupled tobovine serum albumin or KLH via its fatty acid side chain withoutmodifying the crucial phosphoserine moiety.

These data show that rabbit antibodies recognize PS and 100 mol % DOPEbut not PG or PC. The reactivity against pure DOPE, however, seems to beunrelated to the lipids polar head group because reactivity wasabolished when the antigen contained 50 mol % PC (FIG. 3C). Moreover, incontrast to the specific inhibition to PS binding obtained withwater-soluble PS analogs and PS-containing vesicles, GPE and PhoE didnot inhibit antibody binding to DOPE coated plates (FIG. 4B), whereasall vesicles, irrespective of lipid composition, did. Although theseresults suggest that the antibodies do not specifically bind DOPE, theinventor cannot rule out the possibility that some antibodies recognizehexagonal phase structures adopted by DOPE under certain conditions(Rauch et al., 1986; Rauch and Janoff, 1990). The moiety responsible forthe specificity of binding to PS is dependent on the presentation ofboth the serine head group and the glycerophosphate moiety. Indeed,phosphate groups have been shown to be immunogenic and some phospholipidantibodies are partially inhibited by phosphate buffers andphosphorylated nucleotides (Banedi and Alving, 1990; Alving, 1986). Inany event, it is clear that these antibodies are able to recognize PSbut not DOPE in a bilayer membrane because of the specific inhibitionobtained with PS-containing liposomes in the ELISA (FIG. 4C).Furthermore, fluorescence microscopy and flow cytometry analysis showedthat these antibodies did not bind normal RBC even though these cellsexpress ˜20% of their total phosphatidylethanolamine at the cellsurface. On the other hand, RBC became intensely fluorescent uponexpression of PS at the cell surface by Ca²⁺-induced membrane lipidscrambling (Sims et al., 1989; Baldwin et al., 1990; Bevers et al.,1992; Gaffet et al., 1995).

5.2 Example 2 Induction of Autoimmunity for Prevention of Cancer

Cell membranes contain numerous phospholipid species all of which arestructurally and organizationally tightly regulated over the lifespan ofthe cell. A large body of evidence indicates that phosphatidylserine(PS), unlike other phospholipid species, undergoes a dramaticredistribution in the cell's plasma membrane and becomes expressed inthe cell's outer membrane leaflet upon acquisition of a pathologicphenotype. Phenotype-dependent redistribution of PS has been shown tooccur during programmed death cell (apoptosis), platelet activation,cell aging and tumorigenesis. Because PS seems to be a ubiquitous markerof these pathologic cells, it may serve as a specific target epitope foraberrant cell populations and a therapeutic target for cancer treatment.To test the feasibility of this approach for the treatment of cancer, anautoimmune response against PS was raised in mice using the disclosedimmunogens that preserve the lipid's critical head-group and presents PSas a carrier bound hapten.

5.2.1 Synthesis Method B for Prevention of PS-carrier Production

The method is similar to that shown in Example 1 except, instead ofcoupling PS to a sulfhydryl-bearing carrier protein via thiol-disulphideexchange, SPDP-PS is “deblocked” with (tris[2-carboxyethyl]phosphineHCl) to yield a free sulfhydryl which is then directly coupled tomaleimide activated carrier proteins (FIG. 1).

5.2.2 Immunization Against PS Restricts Tumor Growth and Metatasis

To test whether an autoimmune anti-PS response is protective, growth ofseveral transplantable mouse tumors were determined in mice preimmunizedwith lipid antigen.

5.2.2.1 Mouse MBT-2 Murine Bladder Carcinoma

C3H mice were given subcutaneous immunizations with 0.1 ml of saline(0.9% NaCl) or 0.1 ml of BSA-PS immunogen on day 0 with Provax adjuvant(IDEC Pharmaceuticals) and again on day 7 (1 mg/ml BSA-PS insaline:Provax 2:1). On day 14 the mice were injected with 5×10⁴syngeneic murine MTB-2 bladder carcinoma cells into the wall of thebladder. On day 38-40, the mice were necropsied and the bladders wereremoved, weighed, and the presence or absence of tumor was confirmedhistologically (Table 2).

TABLE 2 MOUSE MBT-2 MURINE BLADDER CARCINOMA Individual bladder weights(grams) Control PS immunized 0.155 0.039 0.149 0.048 0.144 0.047 0.1500.046 0.152 0.040 0.155 0.043 0.148 0.047 0.150 0.039 0.151 0.036 mean0.150 0.049 (range) (0.144-0.155) (0.039-0.048) Mann-Whitney U-test:Control vs PS immunized, p < 0.0001

5.2.2.2 RENCA Murine Adenocarcinoma

BALB/c mice were given subcutaneous immunizations with 0.1 ml saline or0.1 ml of KLH-phosphatidylcholine (PC), or 0.1 ml of KLH-PS immunogen onday 0 with Provax adjuvant (IDEC Pharmaceuticals) and again on day 7.Immmunogens were 1 mg/ml in saline:Provax 2:1. On day 14 the mice weregiven intravenous injections of 1.2×10⁴ syngeneic RENCA adenocarcinomacells. On day 38-40, the mice were necropsied for the presence of lungmetastasis. Lungs were placed in Bouin's solution and the individualmetastasis enumerated (Table 3).

TABLE 3 RENCA MURINE ADENOCARCINOMA Individual Number of Lung MetastasisControl KLH-PC immunized KLH-PS immunized 10  8 5 7 8 2 7 5 1 5 5 1 5 41 3 4 0 3 0 1 0 Xmed = 6.0 Xmed = 4.5 Xmed = 1.0 Mann-Whitney U-test:Control vs KLH-PC, N.S. (p > 0.05) Control vs KLH-PS, p > 0.02 KLH-PC vsKLH-PS, p > 0.005

5.3 Example 3 Induction of Autoimmunity for Immunotherapy of Cancer

BALB/c mice were given an intravenous injection of 20,000 RENCA cells onday 0 and therapy was started on day 3, comprising no further treatment(controls), or immunization with the 0.1 ml of PS-BSA preparation (1mg/ml antigen in saline:Provax 2:1). 100 μwas given in two sites thefirst week, followed by two injections of 50 μg in two sites 7 and 14days later. The mice were monitored and lungs were harvested on day 31and the number of individual tumor nodules enumerated (Table 4).

TABLE 4 THERAPY OF EXPERIMENTAL METASTASIS OF MURINE RENALADENOCARCINOMA BY AUTOREACTIVE ANTI-LIPID IMMUNITY Number of LungMetastasis Control PS-BSA Immunized 21 11  14 8 13 5 11 2  9 2 median 135 p < 0.05

5.4 Example 4 Induction of Autoimmunity for Immunotherapy of Leukemia

C57B1/6 mice were inoculated subcutaneously (s.c.) with PS-expressingEG7 lymphoma cells (determined by the ability of the cells to be stainedwith the PS-specific reagent, fluorescein-conjugated annexin V). Themice were sorted into treatment groups (6-8 animal/group) when tumorsranged in size between approximately 75-100 mm³ at which timeimmunization was initiated. The tumor-bearing mice were immunized with asingle injection of Provax on day 8 with 100 μg of PS-KLH or PS-B SAconjugate. Tumor growth delays ranged from 28 days for the PS-BSA groupand 20 days for the PS-KLH group (FIG. 7). Given the fast growth rate ofthis tumor, (tumor volume doubles every 2.4 days) this growth representsa 11.6 (PS-BSA) and 8.5 (PS-KLH) fold decrease in the tumor doublingtime.

5.5 Example 5 Induction of Autoimmunity for the Therapy of CancerUtilizing β₂-glycoprotein I/Lipid Complexes

β₂-glycoprotein I is a 50 kDa serum glycoprotein (Poltz and Kostner,1979; Wurm, 1984) that binds to negatively charged phospholipids. Whileits function is not clear, it has recently been shown that severalautoimmune responses (Galli et al., 1990; McNeil et al, 1990; OoSting etal., 1993; Roubey, 1994) are directed against β₂-glycoprotein I/lipidcomplexes (Schousboe, 1979). Because many cancer cells expressphosphatidylserine on the cell surface, the generation of ananti-complex response may represent substantial breakthroughs in thetreatment of various cancers. To show this, two types of β₂-glycoproteinI/lipid complexes were formed and tested for their therapeutic efficacyin an in vivo cancer therapy model.

5.5.1 Complex Generation

β₂-glycoprotein I was purified from pooled human plasma using previouslypublished procedures (Poltz and Kostner, 1979; Wurm, 1984).

5.5.1.1 Complex I

Microscope slides were coated with 0.9% agarose in 10 mM Tris-HCl pH 7.4to yield a punch hole volume of 20 μL. The center holes were filledβ₂GPI (300 μg/ml) and the surrounding wells with sonicated small lipidvesicles composed of phosphatidylserine/phosphatidylcholine (50/50). Theplates were developed for 24 h and unbound protein and lipid was removedby washing for 24 h in the same buffer. The precipitates containing theprotein/lipid complexes were excised, emulsified with Freund's adjuvantand used as described below.

5.5.1.2 Complex II

Phosphatidylserine/phosphatidylcholine (7/3) was resuspended in 20 mMNaCl to yield a final lipid concentration of 5 mg/mL. The lipidsuspension was mixed with an equal volume of β₂-glycoprotein I (450μg/mL). The suspension was incubated at 4° C. for 2 h and finally mixedwith 0.2 volumes of Provax adjuvant (IDEC Pharmaceuticals, San Diego,Calif.).

5.5.2 Induction of Autoimmunity for Prevention of Cancer UtilizingPS/β₂-glycoprotein

BALB/c mice were immunized two weeks apart with 100 μl of antigensubcutaneously and intradermally. One week later control mice, micetreated with Freund's adjuvant only, and the mice immunized against thePS/β₂-glycoprotein I complex were challenged by an intravenous injectionof 20,000 cultured RENCA cells. The lungs of all were harvested 32 dayslater. Weight of lungs (Table 5) and the number of individual tumornodules (Table 6) were recorded.

TABLE 5 INHIBITION OF THE FORMATION AND GROWTH OF MURINE RENAL CELLADENOCARCINOMA (RENCA) IN THE LUNGS OF SYNGENEIC BALB/c MICE AFTERIMMUNIZATION AGAINST PS-β₂-GLYCOPROTEIN 1 COMPLEX Lung Weights (mg)Adjuvant Control Adjuvant Alone β₂-glycoprotein Complex 404 427 268 356349 241 349 316 220 332 284 191 287 230 185 262 177 229 158 209 150Xmean = 303 +/− 63 Xmean 321 +/− 66 Xmean = 199 +/− 38* Xmedian = 304Xmedian = 316 Xmedian = 188* *p < 0.001

TABLE 6 NUMBER OF LUNG TUMOR NODULES Adjuvant Control Adjuvant Aloneβ₂-glycoprotein Complex 32 35 18 31 21 15 24 19 11 17 18 10 15 14 10 14 8 12  5 11  3 Xmean = 20 +/− 8 Xmean = 21 +/− 7 Xmean = 10 +/− 5Xmedian = 16 Xmedian = 19 Xmedian = 10* *p < 0.02

Taken together, these data demonstrate that the generation of an immuneresponse against the disclosed phosphatidylserine conjugates iseffective for both the prevention and treatment of cancer and that suchtreatment and prevention is independent of tumor type and the tumor'sphysical location in the host. The potential therapeutic and preventiveapplicability in humans is strongly supported by the fact that very hightiters of antibody can be generated in primates.

5.6 Example 5 Animal Models

5.6.1 Materials and Methods

5.6.1.1 Animals

Balb/c mice are purchased from the National Cancer Institute (Frederick,Md.). Animals were maintained in facilities approved by The AmericanAssociation for Accreditation of Laboratory Animal Care, in accordancewith U.S. Department of Agriculture, Department of Health and HumanServices, and NIH regulations and standards.

5.6.1.2 Tumor Cell Cultures

Tumor cells are grown as monolayer cultures in Eagle's MEM with 5% fetalbovine serum, vitamins, pyruvate, L-glutamine and non-essential aminoacids at 37° C.

5.6.1.3 Immunotherapy with PS-hapten-conjugate

Mice are given subcutaneous immunizations with buffer, KLH-PC, or KLH-PS7 days and 14 days after administration of the syngeneic tumor cells.Provax adjuvant is used with all immunogens.

5.6.1.4 Tumor Cell Injections

Cell suspensions are prepared by trypsinization. Intravenous injectionof about 1.2×10⁴ RENCA cells results in ˜10-30 lung tumor nodules in 3-4weeks. The injection of 5×10⁴ MTB-2 cells into the wall of the bladderresults in 100-200 mg tumors in 100% of the mice within 40 days.

5.6.1.5 Therapeutic Efficacy

The end points of therapy are quantitative and allow statisticalcomparison of antitumor effects and measurement of therapeutic benefit.In the RENCA model, the weight of the lungs and the number of lung tumornodules (2-4 mm) are compared between experimental groups. In the MTB-2model, the weights of the bladders and presence of metastasis to theregional lymph nodes are quantified.

5.7 Example 7 Characterization of Autoreactive Immunity

These studies were designed to determine the nature of immune responsethat is responsible for the PS-dependent killing of syngeneic tumormodels. Briefly, the contribution of both cell-mediated and humoralimmune mechanisms were examined.

5.7.1 Assay of Macrophage-mediated Tumor Cell Cytotoxicity

Macrophages are obtained from the peritoneum of appropriate groups ofmice. ¹²⁵I-iododeoxyuridine labeled tumor cells are collected bytrypsinization and plated into microtiter plates at 5×10³ cells/well(macrophage:target cell ratio=25:1). As controls, labeled target cellsare plated without macrophages. After 72 k, the remaining viable targetcells are lysed with detergent. Radiation is measured and the percent ofmacrophage-mediated cytotoxicity is computed.

5.7.2 Assay of Spleen and Lymph-node Cell Cytotoxicity and Inhibition ofTumor Cell Growth

Spleens and lymph nodes are removed from control, tumor-bearing, andimmunized, tumor-bearing mice. Cell suspensions are prepared and 10⁴cells are added to 96-well flat-bottomed microtiter plates withsyngeneic target tumor cells (2×10⁵ lymphocytes/well). Inhibition oftumor growth is measured at 120 h by the hydrolysis of hydroethidine.Cytotoxicity is determined by incubating spleen and lymph node cellswith ¹²⁵I-target cells described above.

5.73 Data Analysis

Differences in the number of metastases between groups is assessed bythe Mann-Whitney test. Significance of cytotoxicity is analyzed byStudents t-test.

5.7.4 Assay of APA (Humorasl Response)

Immunized mice are screened for autoreactive antibody by standardsandwich pan anti-mouse Ig. Organs tested included frozen sections ofstomach, colon, kidney, liver, lungs, spleen, lymph nodes and muscle.APA titers are assessed by anti-phospholipid antibody assay usingstandard clinical laboratory (ELISA) technique.

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All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecomposition, methods and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims. Accordingly, the exclusive rights sought to be patentedare described in the claims below.

What is claimed is:
 1. A method of making an antibody that specificallybinds to phosphatidylserine, said method comprising administering to ananimal a pharmaceutical composition comprising an immunologicallyeffective amount of a phosphatidylserine/polypeptide conjugatecomposition, wherein the phosphatidylserine is covalently coupled to thevolyelptide.
 2. The method of claim 1, wherein a the pharmaceuticalcomposition comprises a phosphatidylserine/BSA, phosphatidylserine/KLH,phosphatidylserine/BGG, or phosphatidylserine/β₂-glycoprotein Iconjugate.
 3. The method of claim 2, wherein said polypeptide isβ₂-glycoprotein I.
 4. The method of claim 1, wherein the antibody islinked to a detectable label.
 5. The method of claim 4, wherein theantibody is linked to a radioactive label, a fluorogernic label, anuclear magnetic spin resonance label, biotin or an enzyme thatgenerates a detectable product upon contact with a chromogenicsubstrate.
 6. The method of claim 4, wherein the antibody is linked toan alkaline phosphatase, hydrogen peroxidase or glucose oxidase enzyme.7. The method of claim 1, wherein the antibody is a monoclonal antibody.8. A method of making an antibody that specifically binds tophosphatidylserine, said method comprising administering to an animal apharmaceutical composition comprising an immunologically effectiveamount of a phosphatidylserine/polypeptide conjugate composition,wherein the phosphatidylserine/polypeptide conjugate composition is nota phosphatidylserine/KLH conjugate composition, and wherein thephosphatidylserine is covalently coupled to the polypeptide.
 9. Themethod of claim 8, wherein the pharmaceutical composition comprises aphosphatidylserine/BSA, phosphatidylserine/BGG, orphosphatidylserine/β₂-glycoprotein I conjugate.
 10. The method of claim9, wherein said polypeptide is β₂-glycoprotein I.
 11. The method ofclaim 8, wherein the antibody is linked to a detectable label.
 12. Themethod of claim 11, wherein the antibody is linked to a radioactivelabel, a fluorogenic label, a nuclear magnetic spin resonance label,biotin or an enzyme that generates a detectable product upon contactwith a chromogenic substrate.
 13. The method of claim 11, wherein theantibody is linked to an alkaline phosphatase, hydrogen peroxidase orglucose oxidase enzyme.
 14. The method of claim 8, wherein the antibodyis a monoclonal antibody.
 15. A method of making a monoclonal antibodythat specifically binds to phosphatidylserine, said method comprisingadministering to an animal a pharmaceutical composition comprising animmunologically effective amount of a phosphatidylserine/polypeptideconjugate composition, wherein the phosphatidylserine is covalentlycoupled to the polypeptide.
 16. The method of claim 15, wherein thepharmaceutical composition comprises a phosphatidylserine/BSA,phosphatidylserine/BGG, or phosphatidylserine/β₂-glycoprotein Iconjugate.
 17. The method of claim 16, wherein said polypeptide isβ₂-glycoprotein I.
 18. The method of claim 15, wherein the antibody islinked to a detectable label.
 19. The method of claim 18, wherein theantibody is linked to a radioactive label, a fluorogenic label, anuclear magnetic spin resonance label, biotin or an enzyme thatgenerates a detectable product upon contact with a chromogenicsubstrate.
 20. The method of claim 18, wherein the antibody is linked toan alkaline phosphatase, hydrogen peroxidase or glucose oxidase enzyme.